Volatile Organic Compounds as In Vitro Biomarkers of Leishmania major

Abstract

Approximately 1 billion people live in leishmaniasis-endemic regions.1 Cutaneous leishmaniasis causes 0.7–1.3 million cases annually, while visceral leishmaniasis affects 50,000-90,000 people annually and is 95% fatal without treatment.1-3 Pediatric cases comprise the majority of reported cases.4-5 Contemporary diagnostics are invasive, costly, or insufficiently sensitive and specific.6-8 Volatile organic compounds (VOCs), which serve to attract insect vectors, represent promising noninvasive biomarkers. We sought to characterize the in vitro VOC profile of Leishmania major to identify compounds with diagnostic potential. Leishmania major Friedlin strain promastigotes were cultured in triplicate in Schneider’s medium with 10% fetal bovine serum. Headspace VOCs were collected on thermal desorption tubes (Markes, UK) and analyzed by comprehensive two-dimensional gas chromatography (Agilent, USA) coupled to time-of-flight mass spectrometry (SepSolve, UK). Dynamic background subtraction, curve-fitting integration, and compound identification were performed in ChromSpace software. Analytes were matched to the NIST library and normalized to internal 1,3-difluorobenzene standards. Differential abundance between parasite and media-alone samples was assessed using Welch’s t-test with Benjamini-Hochberg false-discovery rate (FDR) correction. Gas chromatograms revealed distinct VOC peaks in L. major cultures versus media controls (Figure 1). Mass spectrometry identified 302 unique VOCs, 30 of which showed significantly different normalized abundances after FDR correction (Figure 2). Parasite cultures exhibited enrichment of 3 and depletion of 27 compounds relative to media controls. L. major produces reproducible and statistically distinct VOC profiles under defined culture conditions. To our knowledge, this is the first characterization of Leishmania-associated VOCs generated in vitro. The pattern represents a metabolic fingerprint of parasite activity and highlights candidate biomarkers with diagnostic potential. These findings support further investigation of VOC-based tools for automated culture detection and noninvasive diagnosis in resource-limited settings.